The 31.2% decrease in HIV acquisition reported in the RV144 trial has raised hopes that vaccine protection may be achievable. We have pursued a bimodal vaccine approach to induce both cellular and humoral immunity, our recent rhesus macaques (RM) studies showed that recombinant protein immunogens (SIV Gag-Pol particles, HIV Tat, and multimeric HIV clade C (HIV-C) gp160) achieved complete protection in some RMs against multiple challenges with a heterologous clade C SHIV (SHIV-C). Our study simultaneously linked both cellular and humoral immune responses to protection. In all, we have five vaccine-protected RMs that remained free of persistent, systemic infection; all have high-titer neutralizing antibodies (nAbs) induced by multimeric gp160 against SHIV-C encoding env of a different HIV-C strain with 22.2% divergence compared to the gp160 immunogen. Our overall hypothesis is that vaccine-induced nAb responses can protect against heterologous virus acquisition. We have developed new tools to determine the epitope specificity of protective Abs from polyclonal sera. Our strategy involves a) differential biopannin with recombinant peptide libraries to isolate HIV-C Env mimotopes linked to protection, b) isolation of single B cells specific for a given mimotope/epitope, 3) PCR amplification of RM immunoglobulin variable regions, and 4) generation of recombinant Abs. These novel approaches have led to the isolation of two new chimeric simian/human nmAbs with predicted epitope specificity. We now seek to use these tools for the following Specific Aims: 1. to characterize the epitopes recognized by polyclonal Abs of vaccine-protected RMs by subtractive biopanning. First, we will positively select recombinant phages encodin random peptide libraries by biopanning with sera from a protected RM, followed by negative counter-selection with serum from vaccinated, unprotected RM without significant nAb levels. After several rounds of positive/negative selection, recombinant phages will reflect mimotopes linked to protection. 2. to isolate antigen-specific single B cells from the protected RM and PCR amplify the heavy/light chain variable immunoglobulin regions, using our newly generated RM-specific primers that led to the successful isolation of nmAbs with the predicted specificity as well as potent neutralization. 3. to perform passive immunization in humanized chimeric mice with the novel nmAbs to demonstrate protection against mucosal HIV acquisition. These experiments will show that HIV vaccine-induced nAbs (as opposed to nmAbs isolated from chronically infected individuals) have the potential to prevent HIV acquisition. Our studies will also identify the protective epitopes in vivo and provide important information for HIV/AIDS vaccine design and optimization.